JP2011502535A - Tumor necrosis factor superfamily mRNA expression through Fc receptors in peripheral blood leukocytes - Google Patents

Abstract

  Disclosed are methods for predicting patient responsiveness to treatment of rheumatoid arthritis involving changes in expression of the tumor necrosis factor superfamily (“TNFSF”)-2, TNFSF-8, or TNFSF-15. Also disclosed are methods for monitoring the effectiveness of such treatments. Further disclosed are methods of screening for compounds for use in the treatment of rheumatoid arthritis. Also disclosed is a method for monitoring disease status over time in patients with rheumatoid arthritis.

Description

<Cross-reference of related applications>
This application is a non-provisional application of US Provisional Application No. 61 / 002,967, filed on November 14, 2007, and US Patent Application No. 12 / 296,423, filed October 7, 2008. Partial continuation application. The US patent application No. 12 / 296,423 is a national phase of the international patent application PCT / US2007 / 008559 filed on April 5, 2007, and the international patent application PCT / US2007 / 008559 is 2006. Claims priority based on US Provisional Application No. 60 / 790,511 filed April 7

<Technical field>
The present disclosure provides methods for predicting patient responsiveness to treatment of rheumatoid arthritis related to tumor necrosis factor superfamily members or cytokines, methods for monitoring the effectiveness of such therapies, and rheumatoid arthritis The present invention relates to a method for screening compounds for use in therapy. The present disclosure also relates to a method for monitoring disease status in rheumatoid arthritis patients.

  Autoimmune diseases are characterized by the production of either antibodies that react with host cells or autoreactive immune effector T cells. Autoantibodies are often identified in specific types of autoimmune diseases, such as anti-acetylcholine receptor antibodies in myasthenia gravis and anti-DNA antibodies in systemic lupus erythematosus. However, such autoantibodies are not found in many types of autoimmune diseases. Furthermore, autoantibodies are often detected even in healthy individuals, but such antibodies do not cause autoimmune diseases. Thus, in addition to autoantibodies, clearly further unidentified mechanisms are involved in the pathogenesis of autoimmune diseases.

  Once the autoantibody binds to the target host cell, it is thought that the complement cascade is activated to form a C5-9 membrane attack complex on the target cell membrane that leads to the death of the host cell (Esser, Toxicology 87, 229 (1994)). Chemotaxis factors as by-products such as C3a, C4a, or C5a mobilize additional leukocytes to the lesion (see Hugli, Crit. Rev. Immunol. 1, 321 (1981)). Leukocytes recruited to the lesion or naturally occurring leukocytes recognize antibody-bound cells (immunocomplexes) through Fc receptors (“FcR”). Once FcR is cross-linked by immune complexes, leukocytes release TNF-α (see Devets et al., J Immunol. 141, 1197 (1988)), which binds to specific receptors on the host cell surface. Cause apoptosis or cell damage (see Michelau et al., Cell 114, 181 (2003)). Activated FcR also initiates the release of chemotactic cytokines and recruits different leukocyte subsets to the lesion (see Chantry et al., Eur. J. Immunol. 19, 189 (1989)). This is a general hypothesis about the molecular mechanism of FcR-related autoimmune diseases.

Rheumatoid arthritis (“RA”) is an immune disease with inflammation of the gastrointestinal tract. Despite being well characterized clinically, its etiology is not well understood. RA is usually characterized by persistent inflammatory synovitis involving a symmetric distribution of peripheral joints. This can lead to changes in cartilage destruction, bone erosion, and joint integrity. The cause of RA is not yet known, but the predominance of CD4 + T cells in the synovium, an increase in soluble IL-2 receptors (produced from activated T cells) in the blood and serum of RA patients, and From the marked remission of the disease by the removal of T cells, it is strongly speculated that CD4 + T cells play an important role in this disease. RA is associated with the accumulation of TNF-α (also known as TNFSF-2) in the joint. TNF-α usually serves to move white blood cells to fight infections and other invaders that cause inflammation in the affected area. Healthy bodies can remove excess TNF-α, but the body of rheumatoid arthritis patients cannot. As a result, more white blood cells move to the affected area. This accumulation of TNF-α, particularly in rheumatic joints, causes inflammation, pain, and tissue damage.

The IgG Fc receptor (FcγR) is known to react with immune complexes (IC) (a combination of an epitope and an antibody made against this epitope) that elicits various inflammatory responses. Although specific antigens are not fully characterized, IC is frequently confirmed in joint lesions of RA patients. IC is also known to activate complement cascades that establish inflammation, as well as antibody-dependent cellular cytotoxicity (ADCC) by binding of various leukocytes to FcγR. To date, locally infiltrating leukocytes have been collected from synovial fluid and studied. However, these collections contained both new and depleted cells and the results were difficult to interpret. Since peripheral blood leukocytes play a major role in the pathogenesis of RA as they migrate to the disease site, numerous experiments have been performed to mimic such functions in vitro. Typically, mononuclear leukocytes are separated, suspended in culture medium, and incubated with various stimulants or effector cells in a CO ₂ incubator. However, the conditions under which such assays are performed are physiological conditions due to the lack of communication with different cell populations, oxygen supply from red blood cells, as well as complex interactions with plasma proteins and other components. Is not near. During a long incubation period, a second reaction can occur. Furthermore, due to labor intensive techniques and substantial variation from experiment to experiment, these in vitro tests have little application in diagnostic tests.

  The treatment of RA focuses on analgesia, reducing inflammation, protecting joint structures, maintaining function, and controlling systemic lesions. Options include aspirin and other non-steroidal anti-inflammatory agents; methotrexate, gold compounds, D-penicillamine, antimalarials, and anti-rheumatic agents such as sulfasalazine; glucocorticoids; TNF-α neutralization such as infliximab and etanercept Drugs; and immunosuppressive agents such as azathioprine, leflunomide, cyclosporine, and cyclophosphamide. Since the choice of therapeutic option depends on the assessment of disease status in RA patients, it would be desirable to develop new methods for assessing disease status and monitoring disease progression.

The quantification result of the gene expression of various TNFSF mRNA in the peripheral leukocytes via FcγR is shown. The quantification result of the gene expression of various TNFSF mRNA in the peripheral leukocytes via FcγR is shown. The quantification result of the gene expression of various TNFSF mRNA in the peripheral leukocytes via FcγR is shown. The fold increase in various TNFSF mRNA levels induced by heat-aggregated IgG (HAG) stimulation in the whole blood of rheumatoid arthritis patients and control patients is shown compared to unstimulated whole blood from said patients. The fold increase in various TNFSF mRNA levels induced by heat-aggregated IgG (HAG) stimulation in the whole blood of rheumatoid arthritis patients and control patients is shown compared to unstimulated whole blood from said patients. The fold increase in various TNFSF mRNA levels induced by heat-aggregated IgG (HAG) stimulation in the whole blood of rheumatoid arthritis patients and control patients is shown compared to unstimulated whole blood from said patients. Shown is a comparative fold increase in various TNFSF mRNA levels induced by heat-aggregated IgG (HAG) stimulation in the whole blood of rheumatoid arthritis patients and control patients compared to unstimulated whole blood from said patients. Shown is a comparative fold increase in various TNFSF mRNA levels induced by heat-aggregated IgG (HAG) stimulation in the whole blood of rheumatoid arthritis patients and control patients compared to unstimulated whole blood from said patients. Shown is a comparative fold increase in various TNFSF mRNA levels induced by heat-aggregated IgG (HAG) stimulation in the whole blood of rheumatoid arthritis patients and control patients compared to unstimulated whole blood from said patients.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS The present disclosure provides for the differential mRNA transcription pattern in leukocytes in response to specific cellular stimuli in assessing whether RA patients are suitable candidates for a particular therapy. Regarding use. The present disclosure also relates to the use of such differential transcription patterns in assessing whether a therapy given to RA patients is effective. The present disclosure also relates to the use of such differential transcription patterns in the screening of candidate agents for use in treating RA patients. The present disclosure also relates to the use of such differential transcription patterns in the long-term assessment of patient RA status and in monitoring disease progression.

  As mentioned above, the pathology of RA may be related to the interaction between FcγR and IC in immune cells of RA patients. To further evaluate this possibility, heat-aggregated IgG (HAG, a standard model of IC) was used to stimulate FcγR in human whole blood in both healthy adult controls and RA patients. Other stimulants that can be used are phorbol myristate acetate (PMA), phytohemagglutinin (PHA), wheat germ agglutinin (WGA), concanavalin-A (ConA), lipopolysaccharide (LPS), jacalin, fucoidan, thermal aggregation Includes IgE, thermal aggregation IgA, and thermal aggregation IgM.

  HAG was added directly into heparinized whole blood to assess changes in mRNA levels of various members of the tumor necrosis factor superfamily (TNFSF). Although there are multiple FcγRs such as FcγRI, IIa, IIb, and III (GeneBank UniGene database), HAG acts as a universal stimulus that can react with all FcγR subtypes. Changes in mRNA levels of TNFSF mRNA members (see, eg, the GeneBank UniGene database) due to HAG stimulation were quantified.

As described above, the function through FcγR is the function of four subclasses of IgG Fc region (IgG1-4), multiple classes of FcγR (FcγRIa-c, FcγRIIa-c, and FcγRIIIa-b), and leukocytes having FcγR. It consists of different subsets and various downstream intracellular signal cascades. In addition, these proteins have multiple transcript variants and various genetic polymorphisms. Changes in FcγR function in disease states can occur at any level. However, it may not be realistic to characterize each factor in each individual. The present disclosure provides whole blood as a screening tool to identify individuals who can benefit from various downstream assays, such as individual cell analysis, genotyping of various genes, and variations in intracellular signal cascades. Intended for use (including appendages in ex vivo state). Since IC is known to induce TNFSF-2 (= TNF-α) and TNFSF-15 (= TL1A) mRNA, all tumor necrosis factor superfamily (TNFSF) members were screened by this method.

The method used is as follows. Nucleotide sequences of various TNFSF genes were searched from GenBank's UniGene database. PCR primers for each gene were obtained from Primer Express (Applied Biosystem, Foster City, Calif.) And HYBsimulator (RNAture, Irvine, Calif.) (Mitsuhashi et al., Nature 367, 759 (1994); , 1090 (1996)). A summary of the sequence is shown in Table 1 below. Oligonucleotides were synthesized by IDT (Coralville, Iowa) Tsukuba Oligo Service (Tsukuba, Japan), Nippon Easy Tea (Toyama, Japan) and Hokkaido System Science (Sapporo, Japan).
Table 1. Primer sequence

  Thermoaggregated IgG (HAG) was prepared by heating 20 mg / mL human IgG (Sigma, St. Louis) in PBS at 63 ° C. for 15 minutes (Ostreiko et al., Immunol. Lett. 15, 311 (1987)). reference). 1.4 μl HAG or control (phosphate buffered saline) was added into 8-well strip microtubes and stored at −20 ° C. until use. 70 μl of fresh heparinized whole blood (held at 4 ° C. until stimulation with IC) was added in triplicate to each well, the lid was closed and incubated at 37 ° C. for 4 hours. Blood samples were processed by blood collection on the same day. After treatment, each blood sample was stored frozen at −80 ° C. until use.

mRNA and cDNA are described in Mitsuhashi et al. , Clin. Chem. 52, 634 (2006) and was prepared from whole blood by the following method. The methods disclosed in US patent application Ser. No. 10 / 796,298, incorporated herein by reference, may also be used. Briefly, 50 μl of whole blood was transferred into it to capture leukocytes into a 96-well filter plate. These filter plates were placed on the collection plate and 150 μl of 1.5 mM Tris, pH 7.4 was added. Following 120 × g for 1 minute at 4 ° C., 50 μl of blood sample was added to each well and immediately centrifuged at 120 × g for 2 minutes at 4 ° C., followed by 300 μl of PBS at 4 ° C., 2000 × g for 5 minutes. Washed once by centrifugation. Then 1% 2-mercaptoethanol (Bio Rad, Hercules, CA, USA), 0.5 mg / ml proteinase K (Pierce, Rockford, Illinois, USA), 0.1 mg / ml salmon sperm (5 Prime Eppendorf) / Brinkmann, Westbury, New York, USA), 0.1 mg / ml E.I. 60 μl of lysis buffer stock supplemented with E. coli tRNA (Sigma), each 10 mM specific reverse primer mixture, and standard RNA34 oligonucleotide was added to the filter plate, followed by incubation at 37 ° C. for 10 minutes. The filter plate was then oligo (dT) immobilized microplate (GenePlate, RNAture) (Mitsuhashi et al., Nature 357, 519 (1992); Hamaguchi et al., Clin. Chem. 44, 2256 (1998) (both see And is centrifuged at 4 ° C. and 2000 × g for 5 minutes. After overnight storage at 4 ° C., the microplate was washed 3 times with 100 μl simple lysis buffer followed by 4 × 150 μl wash buffer (0.5 M NaCl, 10 mM Tris, pH 7.4, 1 mM EDTA). Washed 3 times at ° C. For cDNA, add buffer containing 1xRT-buffer, 1.25 mM dNTPs, 4 units of rRNasin, and 80 units of MMLV reverse transcriptase (Promega) (without primer) and incubate at 37 ° C for 2 hours To directly synthesize in 30 μl of each well. In the solution, cDNA stimulated by a specific primer was present, and the cDNA stimulated by oligo (dT) remained fixed in the microplate (Hugli, Crit. Rev. Immunol. 1, 321 (1981). )). For SYBR green PCR (see Morrison et al., Biotechniques 24, 954 (1998) (incorporated herein by reference)), dilute the cDNA 4 times in water and transfer 4 μl of the cDNA solution directly to a 384 well PCR plate. Transfer 5 μl of iTaq SYBR master mix (Bio Rad, Hercules, Calif.) And 1 μl of oligonucleotide mixture (15 μM each forward and reverse primer), and PCR in PRISM 7900HT (ABI) at 95 ° C. One cycle of minutes followed by 45 cycles of 95 ° C. for 30 seconds and 60 ° C. for 1 minute. TaqMan PCR may also be used, in which case the cDNA solution is transferred directly to a 384 well PCR plate and 5 μl TaqMan Universal Master Mix (ABI) and 1 μl oligonucleotide mixture (15 μM forward and reverse primers each, and 3 PCR is performed in PRISM 7900HT (ABI) with one cycle at 95 ° C. for 10 minutes, followed by 45 cycles at 95 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 60-65 ° C. for 1 minute. . Each gene was amplified individually.

  To confirm that no primer dimer was produced under the conditions of SYBR Green PCR, 1 × RT buffer was used as a negative control. In addition, melting curves were analyzed in each case to confirm that the PCR signal was derived from a single PCR product. Analysis software (SDS, ABI) determined the cycle threshold (Ct), which is the number of PCR cycles required for the production of a certain amount of PCR product (fluorescence). In order to calculate ΔCt, the Ct values of the three samples treated with HAG were divided by the average of the Ct values of the PBS control, respectively, and the increase rate of the stimulation sample compared to the unstimulated sample (hereinafter simply referred to as “increase rate”). ) Was calculated as 2 ^ (-ΔCt) by assuming that the efficiency of each PCR cycle was 100%.

1A-1C show mRNA expression via FcγR in peripheral blood leukocytes. Each data point represents the mean + standard deviation (Figure 1A), or mean (Figures 1B, 1C) from triplicate aliquots of whole blood.

  FIG. 1A shows the analysis results of expression kinetics. Three aliquots of 70 μl each of heparinized whole blood were mixed with PBS or 200 μg / ml heat-aggregated IgG (HAG) and incubated at 37 ° C. for 0-8 hours. Thereafter, TNFSF-2 (= TNF-α, ●), TNFSF-8 (▲), or TNFSF-15 (= TL1A, ◆) and β-actin (Δ) mRNA were quantified, and the multiplication factor (y-axis) Was calculated as described above. As shown in FIG. 1A, the induction of TNFSF-2 is very rapid, with a peak around 30 minutes followed by a large persistent peak around 4 hours. In contrast to TNFSF-2, TNFSF-8 (FIG. 1A, ▲) and TNFSF-15 (= TL1A) (FIG. 1A, ♦) increased slowly and the peak was around 4 hours. No housekeeping gene (β-actin) was induced during 8 hours incubation with HAG (FIG. 1A, Δ). Therefore, incubation with HAG to analyze TNFSF-2, -8 and -15 mRNA was fixed at 4 hours. This short incubation (4 hours) is one of the advantages of mRNA-based assays because protein detection to identify drug-induced changes requires at least an overnight incubation.

  FIG. 1B shows a volume response. Heparinized whole blood was incubated with 0-800 μg / ml HAG for 4 hours at 37 ° C., after which various mRNAs were quantified (each symbol is defined as in FIG. 1A). It has been confirmed that TNFSF-2, TNFSF-8 and TNFSF-15 induced by HAG are dose-dependent from 10 μg / ml HAG and saturate at 100-200 μg / ml, but β-actin remains unchanged. It was.

FIG. 1C shows that TNFSF-2 (●), TNFSF-8 (▲), TNFSF-15 (♦) mRNA and external control mRNA (synthetic RNA 34) induced by HAG and collected twice within one week from the same individual. The result obtained by quantifying (◯) is shown. As shown in FIG. 1C, the induction is reproducible, and the r ² value between the first day and the second day among 8 healthy persons is 0.927 (n = 32, p <0.001). (FIG. 1C). The fold increase in externally synthesized RNA added in lysis buffer (RNA34, FIG. 1C, o) is always less than 1.5, suggesting that the assay was performed properly in each experiment.

  The actual blood volume used for mRNA analysis was 50 μl, but this assay used 70 μl of whole blood per reaction (20 μl was reserved during the transfer from the 8-well strip to the filter plate). Therefore, each test consumed as little as 420 μl (70 μl / well × 2 (HAG and PBS) × 3 (3 types)) of whole blood. Thirty different mRNAs were quantified from 50 μl whole blood / well by RT-PCR (90 μl water added to 30 μl cDNA (1: 4 dilution), 4 μl cDNA / PCR). Various TNFSF mRNA measurements were achieved even from 1: 4 dilutions of cDNA. Unlike serum-based tests where serum levels are unpredictable due to variations in hematocrit between individuals, whole blood is easy to manipulate.

Table 2 shows the results of analysis of TNFSF mRNA gene expression via FcR in human leukocytes in peripheral whole blood. The results presented here are expressed as a percentage of “responder” subjects, defined as subjects exhibiting an increase factor greater than 2 by response to HAG stimulation. To compare the frequency of positive reactions between healthy subjects and RA patients for each TNFSF mRNA, a χ ² test was used. Because there were two populations (positive and negative responses), the t-test was applied only to subjects with a fold increase greater than 2.

As shown in Table 2, HAG induced TNFSF-15 (= TL1A) mRNA in all healthy donors and 59 of 61 RA patients (magnification> 2). These results were reproduced in an ex vivo state by a recently published report (Prehn et al., The T cell costimulator TL1A is induced by FcammaR signaling in humanities.
4033 (2007); Cassatella et al. , Soluble
TNF-like cytokine (TL1A) production by immune complexes simulated monocycles in
rheumatoid arthritis, J. et al. Immunol. 178, 7325 (2007) (both incorporated herein by reference). HAG is also known to induce TNFSF-2 (TNF-α) mRNA (Satoh).
et al. , Endogenous production of TNF in
rice with immune complex as a primer, J. et al. Biol. Response Mod. 5, 140 (1986); Chouchakova et al. , Fc gamma RIII-mediated production of TNF-alpha inducibles immune complex independent of CXC chemokin generation, J. Am. Immunol. 166, 5193 (2001) (both incorporated herein by reference). However, as shown in Table 2, TNFSF-2 was found not to be induced in all individuals, and more than half of the subjects did not respond. Furthermore, HAG stimulation induces TNFSF-8 (= CD153, CD30 ligand) and TNFSF-14 (= LIGHT) mRNA in over 1/3 control and RA patients, and in some cases TNFSF-1 (= lymphotoxin α, LTA), TNFSF-3 (= lymphotoxin β, LTB), TNFSF-4 (= CD252, CD134 ligand), TNFSF-6 (= Fas ligand), TNFSF-7 (= CD70, CD27 ligand) and TNFSF-9 However, TNFSF-5 (= CD154, CD40 ligand), TNFSF-12 (= TWEAK), TNFSF-13 (= CD256) and TNFSF-13B (= CD257) were not induced at all or were slightly induced. As shown in FIGS. 1A and 1B, since saturation was achieved in a 4 hour incubation with 200 μg / ml HAG, this individual-to-individual variation, or the presence of responders and non-responders for 4 hours from the start of stimulation Is meaningful. This ex
Vivo assays are expected to be useful as screening platforms for various downstream assays.

Testing for rheumatoid factor (RF) (either type 2 (monoclonal IgM against polyclonal IgG) or type 3 (polyclonal IgM against polyclonal IgG) cryoglobulin) is the standard diagnostic method in cases suspected of RA. The results of TNFSF-2, TNFSF-8, and TNFSF-15 induction by HAG stimulation were classified by RF level to explore the correlation. The results are shown in FIGS. TNFSF-2 (A), TNFSF-8 (B), and TNFSF-15 (C) mRNA induced by HAG were transferred to 40 healthy adult volunteers (control) and RF <30, 30-100, respectively. And quantified from 61 RA patients with> 100 IU / ml. Since two populations were observed (responders with fold increase> 2 and non-responders), the t-test was applied only to the responder population. Analysis by χ ² test did not reveal significant differences between these four groups.

  As shown in FIG. 2, the classification of RA patients by the amount of RF revealed significant differences in TNFSF induction. As shown in FIG. 2C, the fold increase in TNFSF-15 in RA patients with RF> 100 IU / ml is shown for each control (p = 0.01) and RA patients with RF <30 IU / ml (p = 0.04). Significantly less than that. This is probably due to the fact that RF is a natural form of IC and was also present in whole blood when HAG was added ex vivo. The majority of RA patients remained responsive to HAG stimulation. This indicates that, despite prolonged exposure to RF, circulating leukocytes in the peripheral blood of RA patients can still activate IC. The reduced TNFSF-15 response seen in RA patients with high RF may indicate that peripheral blood leukocyte TNFSF-15 receptor function is somehow reduced.

  Considering the activation of FcγR by RF, an evaluation was made as to whether the baseline level of TNFSF-15 mRNA is increased by continuous exposure to RF. As shown in Table 2, TNFSF-5, -13, and -13B mRNA were not induced by HAG, and as a result, by calculating the relative expression of TNFSF-15 compared to these three TNFSF, Measured for Ct. However, TNFSF-15 baseline levels in RA patients were not significantly different from control subjects in all three calculations.

FIG. 3 shows the results shown in FIG. 2 in which TNFSF-2 and -8 (A), TNFSF-2 and -15 (B), and TNFSF-8 and -15 (C) were controlled (◯) and RA (●). ) By comparing with each, the result obtained by converting to xy graph is shown. As shown in FIG. 3C, the magnification of TNFSF-15 was well correlated with that of TNFSF-8 in both RA (●) and healthy subjects (◯), and the r ² values were 0.48 (n = 61, respectively). , P <0.001) and 0.27 (n = 38, p <0.001). However, the increase in TNFSF-8 was low and the population of positive responders was small compared to that of TNFSF-15, so even when RA patients were categorized by RF dose, between control and RA There was no significant difference for TNFSF-8 (FIG. 2B). However, as shown in FIG. 2A, in contrast to TNFSF-15, the increase in TNFSF-2 in RA patients with RF <30 and <100 was significantly greater than that of healthy subjects (p <0.03, 0 .05) It was expensive. The magnification of TNFSF-2 was as follows: TNFSF-8 (FIG. 3A) (control and RA, r ² = 0.03 and 0.02, respectively) and TNFSF-15 (FIG. 3B) (control and RA, respectively, r ² = 0. 11 and 0.003), which is not correlated with TNFSF-2 and TNFSF-8 /
It is suggested that -15 is derived from a different pathway. This was further confirmed by the evidence shown in FIG. 1A that the kinetics of TNFSF-2 and TNFSF-8 / -15 are different.

  TNFSF-2 (= TNF-α) is one of the inflammatory cytokines involved in the pathogenesis of RA, and is present in RA synovial fluid (Saxne et al., Detection of tumor necrosis factor alpha nottator necrosis). factor beta in rheumatoid arthritis synovial fluid and serum, Arthritis Rheum. 31, 1041 (1988), incorporated herein by reference). In situ hybridization has shown that TNF-α transcripts are present in synovial tissue macrophages (MacNaul et al., Analysis of IL-1 and TNF-alpha gene expression in human rhematologic synthesis in situ hybridization, J. Immunol. 145, 4154 (1990), incorporated herein by reference). Furthermore, anti-TNF-α monoclonal antibodies (infliximab, Remicade) and soluble TNF receptors (Etanercept, Enbrel) that block the action of TNF-α have shown clinical efficacy in RA patients (Weaver, The impact of new biologics in the treatment of rhematoid arthritis, Rheumatology (Oxford) 43 Suppl. 3: iii17-iii23 (2004), incorporated herein by reference). Thus, the HAG-induced increase in TNF-α induction seen in RA patients in our ex vivo assay is quite reasonable. Of course, the induction of mRNA does not always correspond to protein synthesis and subsequent biological and clinical consequences due to alternative splicing, post-translational modification, and an inhibitory cascade. However, this ex vivo simulation will be useful as a screening tool for the starting point of various downstream molecular assays, such as genetic polymorphisms of receptors and associated proteins.

  We also compared the clinical characteristics of patients between responders (magnification> = 2) and non-responders (magnification <2) for TNFSF-2 mRNA induced by HAG. The results are shown in Table 3.

  As shown in Table 3, the responder population in the RF <30 IU / ml group was significantly younger than the non-responder population (p = 0.04). There were no significant differences between responders and non-responders in all RF groups for other clinical parameters such as age, sex, duration of disease, CRP, number of swollen joints, and number of joints with tenderness (Table 3). Interestingly, when all patients were combined, the number of patients treated with biological agents such as anti-TNF-α monoclonal antibodies or soluble TNF receptors was significantly higher in the responder population than in the non-responder population. (P = 0.02) (Table 3). The use of these anti-TNF-α agents may increase the effects of TNF-α via FcγR through an unknown negative feedback mechanism, or these patients may be good candidates for biological therapy. RA patients who respond to HAG-induced TNF-α mRNA may further have a significant involvement of TNF-α in RA pathology and may therefore be good candidates for these biological agents There is more sex. Because biological drugs are very expensive, are not effective for all patients, and sometimes show undesirable side effects, the identification of patients who may respond to these drugs has become a personalized medicine It will have clinical utility.

Cytotoxicity tests have generally been used to study actual cell death due to the activity of the immune system believed to occur in RA. Cytotoxicity tests are generally performed by incubating ⁵¹ Cr loaded cells and effector cells at various ratios and quantifying the amount of ⁵¹ Cr radioactivity released from dead or damaged cells (Dunkley et al. al., J. Immunol.Methods 6,
39 (1974)). In some cases, radioactive materials have been replaced with non-radioactive materials, such as fluorometric materials (see Kruger-Kragakes et al., J. Immunol. Methods 156, 1 (1992)), but the basic principle is It has not changed. Thus, cytotoxicity test results reflect actual cell death.

  However, cytotoxicity tests are performed under non-physiological experimental conditions, and it is difficult to evaluate the complex cell-to-cell and cell-to-plasma interactions in such a research process. Furthermore, cytotoxicity tests do not indicate which TNFSF member is responsible for cell death. Since a single effector cell is not sufficient to kill many target cells, once the effector cell recognizes the target cell, the function of the effector cell is not only to kill the target, but also to recruit other effector cells It is also a thing. This mobilization function is thought to be represented by the release of chemotactic factors. The identity of such chemotactic factors released from effector cells will not be revealed by classical cytotoxicity tests. However, the assay system described in this disclosure can simultaneously identify multiple classes of gene expression in effector cells.

  The use of whole blood is preferred over the use of isolated leukocytes in culture. The reason is that the former is more physiological than the latter and can screen the entire population of leukocytes. Longer incubation of whole blood can produce further artifacts. The ideal method is therefore to identify the initial killer and recruitment signals of killer in whole blood during a brief incubation by switching from in vitro to ex vivo. Transcription of mRNA is an earlier event than either protein synthesis or the final biological outcome. Thus, mRNA is a logical target.

  Because this method uses whole blood, it may be used as a diagnostic test for RA to assess possible responsiveness to TNFSF non-activated therapy and monitor therapeutic response. In particular, in a preferred embodiment of a method for determining whether a human having RA is likely to respond to a therapy that targets transcribed mRNA in response to T cell stimulation, as described above, RA Whole blood is collected from the patient and the blood sample is subjected to HAG stimulation and, if necessary, to control stimulation (PBS). The amount of TNFSF-2, TNFSF-8, or TNFSF-15 mRNA may be measured in the sample. RA patients with significantly elevated levels of one or more of these after stimulation with HAG (as indicated, eg, greater than 2 multiplication factors) RA patients are better treated with these mRNAs Is a candidate.

  Further, in a preferred embodiment of the method for assessing in a patient the effectiveness of RA therapy targeting one or more of TNFSF-2, TNFSF-8, or TNFSF-15 mRNA, prior to initiation of treatment, after HAG stimulation The first ratio of the amount of mRNA in the whole blood to the amount after control stimulation is determined. After the start of treatment, a second ratio of the amount of mRNA in whole blood after HAG stimulation to the amount after control stimulation is determined. A significant difference in the ratio such that the first ratio is greater than the second ratio indicates the effectiveness of the treatment. Such treatment may include, for example, administration of infliximab or etanercept.

Importantly, this ex vivo method involves the anti-FcR of one or more of TNFSF-2, TNFSF-8, or TNFSF-15 mRNA, particularly TNFSF-2, which is known to be involved in disease pathology. Can be used to screen for compounds that inhibit the expressed expression. Such compounds would be of interest because these new drug candidates can block mRNA production in leukocytes at the transcriptional level. This will provide a new strategy for drug development against RA.

  In an embodiment of a drug compound screening method using the disclosed system, thereby identifying a putative agent for RA treatment, whole blood is obtained from a responding RA patient, wherein the responder is: An individual who exhibits at least a 2-fold increase in the level of mRNA associated with RA when the leukocytes are exposed to a T cell stimulus such as HAG. The first ratio of the amount of mRNA in the whole blood of the subject after HAG stimulation to the amount after control stimulation was calculated. Additional whole blood samples from subjects were exposed to drug compounds in vitro and then stimulated differentially as described above. Thereafter, a second ratio of the amount of mRNA in the whole blood after HAG stimulation to the amount after control stimulation of these exposed samples was calculated. A significant difference in the ratio, such that the first ratio is greater than the second ratio, indicates that the drug compound is a candidate for further study as a promising treatment for RA.

  In addition, a method for monitoring the disease state of RA patients by measuring the level of one or more of TNFSF-2, TNFSF-8, or TNFSF-15 mRNA in a sample containing leukocytes obtained from a patient is preferred In an embodiment, at a first time, the amount of mRNA in whole blood following T cell stimulation or other stimulation with an in vitro heat-aggregating IgG antibody relative to the amount after control stimulation in vitro. Get the ratio. At a second time following the first time, a second ratio of the amount of mRNA in whole blood after in vitro T cell stimulation to the amount after control stimulation in vitro is obtained. A significant difference in the ratio indicates a change in disease state. For example, disease progression is indicated when the second ratio is greater than the first, while disease regression is indicated when the first ratio is greater.

Claims (32)

A method of determining whether a patient with rheumatoid arthritis is likely to respond to anti-TNF therapy comprising:
Stimulating leukocyte Fc receptors in a first sample from said patient in vitro;
Following stimulation, measuring the amount of mRNA encoding the tumor necrosis factor superfamily (“TNFSF”) protein in the first sample:
Exposing the leukocytes in the second sample to a control stimulus in vitro:
Measuring the amount of said mRNA in a second sample;
Determining the ratio of the amount of mRNA in the first sample to the amount of mRNA in the second sample; and if the ratio is about 2: 1 or greater, the patient may respond to the therapy A method comprising determining to be.   2. The method of claim 1, wherein the mRNA encodes TNFSF-2.   3. The method of claim 2, wherein stimulating leukocytes in the first sample comprises mixing thermally aggregated human IgG with the first sample.   The method of claim 2, wherein at least one of the first and second samples comprises whole blood.   The method of claim 2, wherein the control stimulus is phosphate buffered saline.   3. The method of claim 2, wherein the treatment comprises administration of a drug selected from the group consisting of infliximab and etanercept. A method for determining whether a patient with rheumatoid arthritis is likely to respond to a therapy that inhibits the expression of mRNA encoding a tumor necrosis factor superfamily ("TNFSF") protein via anti-FcR, comprising:
Stimulating leukocyte Fc receptors in a first sample from said patient in vitro;
Following stimulation, measuring the amount of mRNA encoding the tumor necrosis factor superfamily (“TNFSF”) protein in the first sample:
Exposing the leukocytes in the second sample to a control stimulus in vitro:
Measuring the amount of said mRNA in a second sample;
Determining the ratio of the amount of mRNA in the first sample to the amount of mRNA in the second sample; and if the ratio is about 2: 1 or greater, the patient may respond to the therapy A method comprising determining to be.   8. The method of claim 7, wherein the mRNA encodes TNFSF-2.   9. The method of claim 8, wherein the stimulation of leukocytes in the first sample comprises mixing thermally aggregated human IgG with the first sample.   9. The method of claim 8, wherein at least one of the first and second samples comprises whole blood.   9. The method of claim 8, wherein the control stimulus is phosphate buffered saline. A method for identifying a candidate agent for treating rheumatoid arthritis comprising:
First, second, comprising leukocytes from a human having leukocytes that increase transcription of mRNA encoding tumor necrosis factor superfamily ("TNFSF") protein by at least 1.5-fold when exposed to heat-aggregated human IgG, Obtaining a third and fourth sample;
Stimulating leukocyte Fc receptors in the first sample in vitro;
Exposing the leukocytes in the second sample to a control stimulus in vitro:
Measuring the amount of mRNA in the first and second samples after stimulation;
Exposing the third and fourth samples to the drug;
In vitro stimulation of leukocyte Fc receptors in the third sample after exposure;
Exposing the leukocytes in the fourth sample to a control stimulus in vitro:
Measuring mRNA levels in the third and fourth samples after stimulation; and comparing the data obtained from the first sample to the data obtained from the third sample and obtained from the second sample Comparing the data to data obtained from a fourth sample, wherein a significant difference between the comparison data suggests a candidate agent. After measuring the amount of mRNA in the first and second samples, a first ratio of the amount of mRNA in the first sample to the amount of mRNA in the second sample is calculated;
After measuring the mRNA level in the third and fourth samples, a second ratio of the amount of mRNA in the third sample to the amount of mRNA in the fourth sample is calculated; and the first and second ratios are compared, 13. The method of claim 12, wherein the method suggests candidate agents that differ significantly between the ratios.   14. The method of claim 13, wherein the TNFSF protein is TNFSF-2, TNFSF-8, or TNFSF-15.   15. The method of claim 14, wherein stimulating leukocytes in the first and third samples comprises mixing the sample with heat-aggregated human IgG.   15. The method of claim 14, wherein the control stimulus is phosphate buffered saline.   15. The method of claim 14, wherein at least one of the first, second, third, and fourth samples comprises whole blood.   15. The method of claim 14, wherein the significant difference in ratio is that the first ratio is greater than the second ratio.   15. The method of claim 14, wherein the mRNA encodes TNFSF-2.   13. The method of claim 12, wherein the TNFSF protein is TNFSF-2, TNFSF-8, or TNFSF-15.   21. The method of claim 20, wherein stimulating leukocytes in the first and third samples comprises mixing the sample with heat-aggregated human IgG.   21. The method of claim 20, wherein the control stimulus is phosphate buffered saline.   21. The method of claim 20, wherein at least one of the first, second, third, and fourth samples comprises whole blood.   21. The method of claim 20, wherein the mRNA encodes TNFSF-2. A method for assessing the status of a patient's rheumatoid arthritis,
In vitro stimulation of leukocyte Fc receptors in a first sample containing leukocytes and obtained at a first time from a patient;
Measuring the amount of mRNA encoding the tumor necrosis factor superfamily (“TNFSF”) protein in the first sample after stimulation;
Exposing leukocytes in a second sample containing leukocytes obtained from said patient at said first time in vitro to a control stimulus;
Measuring the amount of mRNA in the second sample after stimulation;
Determining a first ratio of the amount of mRNA in the first sample to the amount of mRNA in the second sample;
In vitro stimulation of leukocyte Fc receptors in a third sample containing leukocytes and obtained from a patient at a second time following said first time;
Measuring the amount of mRNA in the third sample after stimulation;
Exposing the leukocytes in the fourth sample containing leukocytes obtained from the patient at the second time in vitro to a control stimulus;
Measuring the amount of mRNA in the fourth sample after stimulation;
Determining a second ratio of the amount of mRNA in the third sample to the amount of mRNA in the fourth sample; and comparing the first and second ratios, a significant difference in the first and second ratios being A method comprising suggesting a change in state.   26. The method of claim 25, wherein the TNFSF protein is TNFSF-2, TNFSF-8, or TNFSF-15.   27. The method of claim 26, wherein stimulating leukocytes in the first and third samples comprises mixing human heat-aggregated IgG with the samples.   27. The method of claim 26, wherein the control stimulus is phosphate buffered saline.   27. The method of claim 26, wherein at least one of the first, second, third, and fourth samples comprises whole blood.   27. The method of claim 26, wherein the second ratio is significantly different in the ratio than the first ratio, and the change in disease state is disease progression.   27. The method of claim 26, wherein the first ratio is significantly greater in the ratio than the second ratio, and the change in disease state is disease regression.   27. The method of claim 26, wherein the mRNA encodes TNFSF-2.

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